Synthesis of ribonucleotides from the corresponding ribonucleosides under plausible prebiotic conditions within self-assembled supramolecular structures

Article abstract of DOI:10.1039/c9nj05601g

Abiotic synthesis of ribonucleotides, mainly at the 5′ position, from the corresponding ribonucleosides within self-assembled supramolecular structures, based on guanosine:borate hydrogels, was carried out in the temperature range of 70-90 °C, using urea and a phosphate source (K₂HPO₄ or hydroxyapatite). Phosphorylation is possible at initial concentrations of guanosine lower than 20 mM and it is more efficient using wet/dry cycles. Monoamidophosphate (and, eventually, diamidophosphate), diamidodiphosphate and pyrophosphate are intermediates in the synthesis of ribonucleotides. These conclusions are supported by NMR spectroscopy and mass spectrometry analysis of samples. On the other hand, after reaction, hydrogels can produce globular aggregates by the addition of water and decreasing temperature, thus confirming that ribonucleotides, once activated under suitable conditions, could form polyribonucleotides.

Full text of DOI:10.1039/c9nj05601g

View Article Online
View Journal
NJC
New Journal of Chemistry
Accepted Manuscript
A journal for new directions in chemistry
This article can be cited before page numbers have been issued, to do this please use: J. A. L. D. Silva, A.
Franco, J. R. Ascenso and L. Ilharco, New J. Chem., 2020, DOI: 10.1039/C9NJ05601G.
Volume 42
This is an Accepted Manuscript, which has been through the
Number
6
21 March 2018
Pages 3963-4776
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
New Journal of Chemistry
rsc.li/njc
A journal for new directions in chemistry
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1144-2546
PAPER
Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
prepared via a microwave-assisted hydrothermal route using
H2O2 and KMnO4 as oxidizing agents
rsc.li/njc

Please do not adjust margins
Page 1 of 4
New Journal of Chemistry
1
2
3
4
5
6
7
View Article Online
DOI: 10.1039/C9NJ05601G
COMMUNICATION
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of Ribonucleotides from the Corresponding
Ribonucleosides under Plausible Prebiotic Conditions within Self-
assembled Supramolecular Structures
A.Francoa, J.R. Ascensoa, L. Ilharcob and J.A.L. da Silvaa*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Abstract: Abiotic synthesis of ribonucleotides, mainly at the 5’ prebiotically plausible reagent, such as borate, since it stabilises
position, from the corresponding ribonucleosides within self- ribose (constituent of the framework of ribonucleosides) in those
assembled
supramolecular
structures,
based
on positions.17 Guanosine forms diesters with borate, leading to the
guanosine:borate hydrogels, were carried out in the range 70 ºC- self-assembly of supramolecular hydrogels, based on stacked
90 ºC, using yet urea and a source of phosphate (K2HPO4 or hydrogen-bonded guanosine quartets, which have high thermal
hydroxyapatite). Phosphorylation is possible at initial stability.18,19 Their structure is related with BBS (Boron Bound
concentrations of guanosine lower than 20 mM and it is more Species, i.e., ribose borate esters),1Error! Bookmark not defined. with only
efficient using wet/dry cycles. Monoamidophosphate (and, one free hydroxyl group (C5’). However, direct phosphorylation of
eventually
diamidophosphate),
diamidodiphosphate
and nucleosides is not effective without an activator, and urea has been
pyrophosphate are intermediates in the synthesis of suggested as a plausible reagent.11,20 The occurrence of urea in the
ribonucleotides. These conclusions are supported by NMR interstellar space21 supports its involvement in prebiotic reactions,
spectroscopy and mass spectrometry analysis of samples. On since it could have been delivered to Earth.22
the other hand, after reaction, hydrogels can produce globular Herein is reported phosphorylation, under prebiologically plausible
aggregates by addition of water and decreasing temperature, conditions, of ribonucleosides, within self-assembled structures
thus confining ribonucleotides that, once activated under (guanosine-borate hydrogels), see Scheme 1. These conclusions are
suitable conditions, could form polyribonucleotides.
supported by NMR spectroscopy and mass spectrometry analysis of
samples based on reactions with guanosine supramolecular
hydrogels or solutions containing urea and phosphate.
Phosphorylation of ribonucleosides is a key step for prebiological
evolution by “RNA world hypothesis”.1 This challenge began at least
in the sixties of the last century.2 Some methods (in different
conditions, e.g., aqueous solutions and dry environments) using
diverse sources of phosphate have been suggested for all
ribonucleosides, leading to phosphorylation at different carbon
atoms and, typically, with higher yields to uridine.3-14 However, in
all the cases, the synthesis of more complex prebiological molecules
would be very limited because the products could be easily
dispersed. This might be overcome with supramolecular hydrogels
as reaction matrix, since they could confine prebiotic molecules.15,16
Phosphorylation of a ribonucleoside at C5’ position is not very
efficient, due to competition of the two other free –OH groups of
the organic molecule. However, by protection of positions C2’ and
C3’ of ribonucleoside, this reaction could be improved with a
Base = Guanine, Adenine, Cytosine
Base
O
HO
Base
1'
5'
H
O
HO
P
O
5'
H
4'
H
1'
H
Phosphate
Urea
4'
O
H
H
H
3' 2'
OH
+ other products
O
O
H
3' 2'
B
OH OH
HO
OH
Guanosine is a building block
for supramolecular hydrogels
Main product confined in hydrogel
Scheme 1 – Phosphorylation of ribonucleosides under plausible prebiotic
conditions.
Phosphorus signals of ribonucleotides by 31P NMR spectroscopy
exhibit chemical shifts at ca. 4 ppm.23 Figure 1 is a 1H-31P HMBC
spectrum24 from reaction of a hydrogel of guanosine with borate
containing urea and K2HPO4, showing phosphorylation of the
nucleoside, mainly in position C5’ (31P signal at 3.90 ppm has cross-
peaks at 3.78 ppm, which are assigned to the protons of the CH2
group, unique of nucleotide). Additional 31P signals at 3.49 and
4.02 ppm with cross-peaks at 4.89 and 4.58, respectively, are
assigned to phosphorylation in positions C2’ and C3’, in this order.
a Centro de Química Estrutural, Instituto Superior Técnico,
Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
b IBB, Instituto de Bioengenharia
e Biociências, Instituto Superior
Técnico, Universidade de Lisboa, Av. RoviscoPais, 1049-001 Lisboa,
Portugal
Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 2 of 4
COMMUNICATION
Journal Name
1
2
3
4
5
This should result from borate that reduces phosphorylation in
positions C2’ and C3’, and simultaneously induces the stability of
the hydrogel.
View Article Online
DOI: 10.1039/C9NJ05601G
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3 - 31P NMR spectra of guanosine hydrogels after (from bottom to
top): 1, 2, 4 and 10 wet/dry cycles of reaction at 90 oC with K2HPO4 and urea,
run in a 400 MHz spectrophotometer; (a) guanosine-3’-phosphate, (b)
guanosine-5’-phosphate and (c) guanosine-2’-phosphate.
Figure 1 - 1H-31P HMBC spectrum of a guanosine hydrogel with borate after 4
wet/dry cycles (2 days of reaction) at 90 oC with K2HPO4 and urea run in a
400 MHz spectrophotometer.
On the other hand, samples with two daily additions of water and
without any cycles have chemical shifts at 3.72 ppm and -6.33 ppm,
assigned to guanosine-5’-phosphate and pyrophosphate signals,
respectively, while for the sample without dryness period the
former signal is absent (see Figure 4). Therefore, reaction
conditions with wet/dry cycles are more efficient than just under
dry conditions. This is in agreement with a recent publication.26
A similar reaction was carried out for a longer period (12 cycles, i.e.
6 days) with a yield of 5% to 5’-ribonucleotide (major species
formed) determined by 31P NMR spectroscopy (see Figure 2 and
Experimental Section in Electronic Supplementary Information).
This yield is significant compared with previous results, since our
reactions typically need lower concentrations and number of
reagents, i.e. they are better adjusted to the prebiological
environments.3-14
Figure 4 - 31P NMR spectra of guanosine hydrogels after (from bottom to
top): 10 wet/dry cycles, dryness and wetness, respectively, for an equivalent
amount of time (5 days), at 90 oC with K2HPO4 and urea, run in a 400 MHz
spectrophotometer. Signals were normalised with (b), inorganic phosphate;
(a) guanosine-5’-phosphate and (c) pyrophosphate.
Urea is also required for activation of phosphate, since the reaction
of a guanosine hydrogel with K2HPO4 in the absence of urea does
not lead to the phosphorylation of guanosine (spectrum not
presented). This suggests the formation of activated species
(required to phosphorylation of nucleosides) from the reaction of
urea and phosphate (see Figure ESI2). In Figure 4 (wet/dry cycles
and dry conditions) another phosphorus species is presented,
pyrophosphate, as it was previously observed.27 This species was
Figure 2 - 31P NMR spectrum of a guanosine hydrogel after 12 wet/dry cycles
of reaction at 90 oC with K2HPO4 and urea, run in
a 400 MHz
spectrophotometer; Cr(acac)3 and D3PO4 were relaxation agent and internal
standard, respectively; (a) guanosine-5’-phosphate, (b) inorganic phosphate,
(c) internal standard (D3PO4) and (d) pyrophosphate.
The level of phosphorylation increases with the number of wet/dry
cycles of reaction, Figure 3. Concomitantly, a structural change on
the hydrogel occurs. In fact, the 11B NMR spectra in Figure ESI1
show that the boron bonds are altered during reaction, suggesting
that guanosine-borate monoesters become more probable than
diesters. This could be due to the decrease of the stability constants
of borate bound to nucleosides derivatives with phosphorylation.25
also detected by ESI-MS(-) of
a solution prepared after a
phosphorylation reaction by water addition and adjusted at pH 1,
with a m/z 177 [pyrophosphate-H], and confirmed by MS/MS
-
fragments 159 [P2O5OH-] and 79 [PO3 ] (see Figure ESI3).28
Pyrophosphate formation was previously detected in different
reaction conditions,29 including at a similar temperature to our
highest value, under more acidic conditions and with nitrogen-rich
compounds.30 Herein, the synthesis of activated phosphate species
is also detected at a lower temperature, i.e. 50 oC (see Figure ESI4).
On the other hand, in the 31P NMR spectrum (not shown) of urea
and K2HPO4 without heating, the signal corresponding to
pyrophosphate was absent. Pyrophosphate was proposed as a
phosphorylation reagent29,30 and it is now suggested as an
intermediate in phosphorylation of guanosine.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 4
New Journal of Chemistry
Please do not adjust margins
Journal Name
COMMUNICATION
1
2
3
4
5
6
7
8
9
Pyrophosphate
formation
has
as
intermediate occurring phosphate minerals.32 However, using hydroxyapatite as
View Article Online
DOI: 10.1039/C9NJ05601G
monoamidophosphate. This species was detected by ESI-MS(-), m/z source of phosphate for phosphorylation, solubilisation of
96 [monoamidophosphate-H] (see Figure ESI5) and by 31P NMR phosphate was detected by 31P NMR spectroscopy, as well as traces
spectroscopy ca. 8.8 ppm (see Figure ESI6) that is according with a of ribonucleotide, see Figure ESI9, after 15 days (30 cycles) of
recent publication.31 On the other hand, diamidophosphate was reaction, despite the low solubility of hydroxyapatite34 at pH of
suggested as a prebiological phosphorylation reagent, although its reaction, i.e., 1000-10000 lower than the concentration in the
synthesis was unlikely from a prebiotic standpoint;27 the synthesis reactions with K2HPO4. This suggests that hydroxyapatite as source
of this species was recently proposed31 using other phosphorus for phosphorylation of ribonucleosides could have occurred in a
compounds as starting material, although they are much rarer on short period as compared with the overall prebiological era.
Earth’s crust than phosphate salts.32 In an aqueous solution (pH 8) Finally, we observed that hydrogels with guanosine and borate,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
prepared from
a solid-state reaction of K2HPO4 and urea, upon heating, followed by water addition and temperature
characterised by ESI-MS(-)(not shown) a m/z 95 corresponding to decrease, form globular aggregates (see Figure ESI10). Generally,
diamidophosphate was identified (not detected by 31P NMR covalently bound hydrogels are quite robust against temperature
spectroscopy, since the detection limit of this technique is higher fluctuations.35 However, guanosine-borate hydrogels stabilised by
than by mass spectrometry), suggesting that in absence of a K+ are non-covalent, since the guanosine-borate esters self-
substrate to phosphorylate (or, probably, with a higher ratio assemble into G4 sticky nanowires by hydrogen bonding and -
urea:phosphate) this species could be formed. Additionally, the 31P stacking, and the wires entangle to produce a fibrous network.36
NMR spectrum of phosphorylation reaction with a guanosine Guanosine:borate mono or diesters may react, when temperature
hydrogel after 1 wet/dry cycle (Figure ESI7) has another signal rises.37 For instance, N1 could no longer be an H donor, affecting
(-3.43 ppm) assigned to diamidodiphosphate,27 another the stability of the G4-quartets. Thus, the gel network could be
intermediate in the pyrophosphate formation. Our results and also partially altered by heating. An alternative explanation for the
from27suggest that upon reaction of urea with phosphate under dry partial destruction of the guanosine:borate hydrogels upon heating
conditions
monoamidophosphate
(and,
eventually, and adding water could be the reversibility of covalent bonds, such
diamidophosphate) is formed, which, by reaction with another as by the borate ester hydrolysisError! Bookmark not
phosphate molecule, leads to diamidodiphosphate. This is defined. (compare with Figure ESI1).
hydrolysed to pyrophosphate, even with low amounts of water. The How could guanosine:borate hydrogels have formed under
formation of activated phosphorus species suggests that prebiotic conditions? The mixture of aqueous solutions of
phosphorylation could have happened as a continuous process nucleosides with potassium and borate minerals (e.g. kaliborite or
during the prebiological period in at least the temperature range santite) and subsequent water evaporation until the formation of
70 oC – 90 oC without degradation of nucleosides or nucleotides. hydrogels could be the answer. These hydrogels are stable (over a
Their confinement into hydrogels with high thermal stability would significant range of temperatures) and, in case of dryness, their
promote the reaction.
properties could be recovered in the presence of water by self-
Other ribonucleosides were included into guanosine hydrogels (see assembly, as consequence of their supramolecular characteristics.
Experimental Section in Electronic Supplementary Information). This behaviour facilitates wet/dry cycles towards phosphorylation in
Subsequently, hydrogels with guanosine/adenosine, and the presence of a suitable and activated phosphate source. This
guanosine/cytidine were tested concerning phosphorylation. All reaction is possible at temperatures, in the range of 70 - 90 oC or
hydrogels require guanosine. In its absence gelation is not achieved, probably at lower temperatures (in this case, with a higher number
since self-assembling is supported by hydrogen bonding18 that is of wet/dry cycles of reaction, compare with Figure ESI4), would lead
stronger to guanosine (from its additional groups with this capacity) to the formation of globules containing ribonucleotides that, once
than to the other ribonucleosides. Figure ESI8 and mass activated, might have led to polyribonucleotides. Moreover, these
spectrometry data show that other ribonucleosides are also gel particles have external boundaries that could promote reactions
phosphorylated. Samples from these reactions solubilised in water at the external or internal surfaces and thus the prebiological
and pH adjusted to 4 (to avoid gelation) with HCl were analysed by evolution.
ESI-MS(-). The mass spectra were very complex, but m/z 322
[cytidine monophosphate-H], 346 [adenosine monophosphate-H] Acknowledgments
and 362 [guanosine monophosphate-H] are only detected in This work has been carried out with financial aid of the FCT
samples containing as starting material cytidine, adenosine and (Portugal)
(Project
numbers
UIDB/00100/2020
and
guanosine, respectively. An amplified effect on the phosphorylation UIDB/04565/2020). The NMR spectrometers are part of the
is observed by the presence of another ribonucleoside in the National NMR Network (PTNMR) and are partially supported by
hydrogel, Figure ESI8, with special emphasis to cytidine Infrastructure Project No 022161.The authors would like to express
(complementary base pair of guanosine).
Minerals containing a source of phosphorus would have been concerning quantification of guanosine-5’-phosphate and 2D
reagents of prebiological phosphorylations. However, some spectrum, to Ana Dias by mass spectrometer analysis, and to Dr.
their gratitude to Dr. Maria João Ferreira for her suggestions
minerals
containing
hydroxidotrioxidophosphate(2−) Hermínio Diogo by the amplified photos of hydrogels.
(hydrogenphosphate) are typically from biological material.33
Prebiological synthesis of crucial molecules for the prebiotic
evolution was hindered by the low solubility product of naturally
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 4 of 4
COMMUNICATION
Journal Name
1
2
3
4
5
6
7
8
9
27- C. Gibard, S. Bhowmik, M. Karki, E. K. Kim and R. KVriieswhnAratimcleuOrtnhlinye,
Nat. Chem., 2018, 10, 212.
28- D. Strzelecka, S. Chmielinski, S. Bednarek, J. Jemielity and J.
Kowalska, Sci. Rep., 2017, 7, 8931.
29- A. D. Keefe and S. L. Miller, J. M o l . Evol. , 1995, 41, 693.
30- R. Osterberg and L .E. Orgel, J. M o l . Evol. , 1972, 1, 241.
31- C. Gibard, E. I. Jiménez, T. Kee, R. Krishnamurthy and M. Pasek,
Angew. Chem. Int. Ed., 2019, 58, 1; Angew.Chem., 2019, 131, 1.
32- M. A. Pasek, M. Gull and B. Herschy, Chem. Geol., 2017, 475,
149.
33- B. P. Onac and D. S. Veres, Eur. J. Mineral., 2003, 15, 741.
34- B. Puzio, M. Manecki and M. Kwaśniak-Kominek, Minerals,
2018, 8, 281.
DOI: 10.1039/C9NJ05601G
Conflicts of interest
There are no conflicts to declare.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Notes and references
1- L. E. Orgel, Crit. Rev. Biochem. Mol. Biol., 2004, 39, 99.
2- C. Ponnamperuma, C. Sagan and R. Mariner, Nature, 1963, 199,
222.
3- C. Ponnamperuma and C. R. Mack, Science, 1965, 148, 1221.
4- G. Steinman, R. M. Lemmon and M. Calvin, Science, 1965, 147,
1574.
5- G. Steinman., D. H. Kenyon and M. Calvin, Nature, 1965, 206,
707.
6- A. Beck, R. Lohrmann and L. E. Orgel, Science, 1967, 157, 952.
7- A. Schwartz and C. Ponnamperuma, Nature, 1968, 218, 443.
8- J. Rabinowitz, S. Chang and C. Ponnamperuma, Nature, 1968,
218, 442.
9- R. Lohrmann and L. E. Orgel, Science, 1968, 161, 64.
10- A. W. Schwartz, Chem. Comm., 1969, 1393.
11- R. Lohrmann and L. E. Orgel, Science, 1971, 171, 490.
12- B. Burcar, M. Pasek, M. Gull, B. J. Cafferty, F. Velasco, N. V. Hud
and C. Menor-Salván, Angew. Chem. Int. Ed., 2016, 55, 13249;
Angew.Chem., 2016, 128, 13443.
13- H. J. Kim, Y. Furukawa, T. Kakegawa, A. Bita, R. Scorei and S. A.
Benner, Angew. Chem. Int. Ed., 2016, 55, 15816; Angew. Chem.,
2016, 128, 16048.
14- N. Kitadai and S. Maruyama, Geosci. Front., 2018, 9, 1117.
15- J. T. Trevors and G. H. Pollack, Prog. Biophys. Mol. Biol., 2005,
89, 1.
16- J. A. L. da Silva and N. G. Holm, J. Colloid Interface Sci., 2014,
431, 250.
17- A. F. Amaral, M. M. Marques, J. A. L. da Silva and J. J. R. Fraústo
da Silva, New J. Chem., 2008, 32, 2043.
18- G. M. Peters, L. P. Skala, T. N. Plank, B. J. Hyman, G. N. M.
Reddy, A. Marsh, S. P. Brown and J. T. Davis, J. Am. Chem. Soc.,
2014, 136, 12596.
19- G. M. Peters, L. P. Skala, T. N. Plank, H. Oh, G. N. M. Reddy, A.
Marsh, S. P. Brown and J. T. Davis, J. Am. Chem. Soc., 2015, 137,
5819.
20- C. Fernández-García, A. J. Coggins and M. W. Powner, Life, 2017,
7, 31.
21- I. Fourré, L. Rosset, H. Chevreau and Y. Ellinger, A&A, 2016, 589,
A18.
22- M. Forstel, P. Maksyutenko, B. M. Jones, B. J. Sun, A. H. H.
Chang and R. I. Kaiser, Chem. Comm., 2016, 52, 741.
23- B. J. Cade-Menun, Geoderma, 2015, 257-258, 102.
24- A. Akiva-Tal, S. Kababya, Y. S. Balazs, L. Glazer, A. Berman, A.
Sagi and A. Schmidt, Proc. Natl. Acad. Sci. USA, 2011, 108, 14763.
25- D. H. Kim, K. F. Faull, A. J. Norris and C. D. Eckhert, J. Mass
Spectrom., 2004, 39, 743.
35- F. Picchioni and H. Muljana, Gels, 2018, 4, 21.
36- J. Zhang, Y. Hu and Y. Li, Gel Chemistry – Interactions, Structures
and Properties, Springer, Singapore, 2018.
37- T. N. Plank and J. T. Davis, Chem Comm., 2016, 52, 5037.
26- S. Becker, C. Schneider, H. Okamura, A. Crisp, T. Amatov, M.
Dejmek and T. Carell, Nat. Commun., 2018, 9, 163.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins